Organoids are three-dimensional multicellular structures derived from stem cells or progenitor cells that self-organize into tissue-like architectures, faithfully recapitulating the structural and functional features of native organs. Matrix Gel serves as the essential scaffold for organoid formation by providing a physiologically relevant extracellular matrix (ECM) environment rich in laminin, collagen IV, entactin, and growth factors such as EGF, FGF, and IGF. These components support cell adhesion, polarization, and differentiation, enabling long-term culture and expansion of organoids derived from various tissues including intestine, liver, brain, lung, and tumor samples.
Matrix Gel–based organoid systems have become indispensable tools in developmental biology, regenerative medicine, drug screening, and precision oncology. Compared with conventional 2D cultures, organoid models allow more accurate investigation of cell–cell and cell–matrix interactions, disease mechanisms, and therapeutic responses.
I. Protocol for Mouse Intestinal Organoids
1. Sample Preparation: Euthanize mouse by cervical dislocation and disinfect the surface with alcohol. Under sterile conditions, excise 3–15 cm of small intestine from the region near the stomach. Carefully remove mesentery and fat from the outer surface using forceps and place the tissue into pre-chilled DPBS containing 1% antibiotics at 4 °C.
2. Sample Washing: Flush the lumen 2–3 times with a syringe. Open the intestine with scissors, place the luminal side up, and gently scrape off villi using a surgical blade. When villi are completely removed (tissue appears translucent), transfer the tissue to a new dish containing DPBS and wash 2–3 times.
3. Initial Processing: Cut the washed intestinal tissue into ~2 mm fragments and transfer to a new 50 mL conical tube. Wash gently 3–5 times with DPBS to remove villus cells and floating fat.
4. Tissue Digestion: Add 10–15 mL of pre-chilled DPBS containing 3–5 mM EDTA to the intestinal fragments and incubate at 4 °C for ~30 min, gently shaking the tube every 10 min.
5. After digestion, discard the EDTA supernatant and gently rinse the tissue 2–3 times with fresh DPBS to remove residual EDTA.
6. Add 10–15 mL of pre-chilled DPBS containing 0.1% BSA to the intestinal fragments. Pipette repeatedly to dissociate crypts from the basement membrane. Examine a small aliquot under a microscope; stop pipetting when abundant crypt-like structures are observed. Filter the suspension through a 70 μm cell strainer and collect the flow-through.
7. Repeat steps 5–6 twice, then centrifuge at 1500 rpm, 4 °C for 3 min.
8. Mixture Preparation: Resuspend the crypt pellet in CeturegelTM Matrix Gel. Use approximately 200–600 crypts per 10 μL of Matrix Gel. Keep the suspension on ice and proceed quickly to prevent premature gelation.
[Note]: The Matrix Gel dilution ratio should be ≥50% to ensure structural stability during culture.
9. Seed 30–50 μL of the mixture into the center of each well of a 24-well plate, avoiding contact with the well walls.
10. Place the plate in a 37 °C CO₂ incubator and incubate for ~30 min to allow gelation.
11. After complete gelation of CeturegelTM Matrix Gel, slowly add pre-prepared intestinal organoid culture medium along the wall of each well (800 μL/well).
12. Culture the 24-well plate in a 37 °C CO₂ incubator. Replace with fresh medium every 3 days and monitor organoid growth. Mouse intestinal organoids typically form within 5–7 days.
Experimental Results
Figure 1. In vitro culture results of mouse small intestinal organoids
II. Protocol for Tumor Organoids
1. Pre-experiment Preparation:
Thaw tumor organoid culture kit components and matrix gel at 4°C.
Prepare sufficient volumes of DPBS containing 1% penicillin-streptomycin (dual antibiotics) and DPBS containing 0.1% BSA.
Prepare one-fifth strength trypsin solution: mix 32 mL basal medium with 8 mL trypsin, pre-warm at 37°C for later use.
2. Sampling:
Under sterile conditions, excise approximately 2 cm of viable tumor tissue near the gastric end. Use forceps to remove surrounding connective tissues, fat, fascia, etc., and place the tissue into ice-cold DPBS (supplemented with dual antibiotics) or culture medium for transport.
3. Washing:
Using surgical scissors, mince the tumor tissue into ~2 mm fragments. Transfer the minced tissue pieces into a new 50 mL conical tube containing DPBS and wash until the supernatant becomes clear. Discard supernatant and add Type I collagenase (~3× volume of tumor tissue). Digest for 30 min, followed by two washes with basal medium.
1. Transfer the collagenase-digested tumor tissue into basal medium containing one-fifth strength trypsin. Incubate at 37°C for 20–30 min. After digestion, add an appropriate volume of basal medium containing 0.1% BSA to neutralize the reaction. Gently mix and collect the supernatant.
2. Add 30 mL of basal medium containing 0.1% BSA to the remaining pellet. Resuspend and triturate the tissue fragments using a 10 mL pipette in a 50 mL conical tube—repeatedly pass the tissue through the pipette tip to generate mechanical shearing forces that dissociate tumor cells from the basement membrane. Repeat 6–10 times. Take a small aliquot for microscopic examination. Stop trituration when abundant single-cell structures are observed. Filter the suspension through a 70 μm cell strainer.
3. Collect the flow-through fraction from filtration. Centrifuge at 300 × g, 4°C for 3 min.
Discard supernatant. Resuspend the pellet in 1 mL of basal medium containing 0.1% BSA. Take 20 μL for microscopy and cell counting. After counting, transfer the desired number of cells into a new tube, centrifuge again at 300 × g for 3 min, discard supernatant, and keep on ice.
4. Resuspend the cancer cell pellet in an appropriate volume of organoid-specific growth medium. Recommended resuspension density: 1–2 × 10⁶ cells/mL. Keep the tube on ice after resuspension. Resuspension time should not exceed 30 seconds to prevent premature gelation of matrix gel.
[Note]: The final dilution ratio of matrix gel should remain above 50% to ensure structural stability during culture.
8.Deposit the mixture of matrix gel and cell suspension as ~30 μL droplets in the center of each well of a 24-well plate. Avoid contact between droplets and the sidewalls of the wells.
[Note]: To prevent premature solidification at room temperature, this step must be performed quickly.
9.Place the inoculated plate in a 37°C CO₂ incubator and incubate for 3–5 min. Then invert the plate and incubate for another 10–20 min to allow complete polymerization of the matrix gel. Once fully solidified, slowly add pre-warmed complete tumor organoid medium along the side of each well—750 μL per well for 24-well plates. Avoid disrupting the already formed gel structure during medium addition.
10.Culture the 24-well plate in a 37°C CO₂ incubator. Change the medium every 3 days, taking care not to disturb the matrix gel. Monitor organoid growth closely. Under optimal conditions, tumor organoids should form within 5 to 10 days.
Figure 2. Cervical cancer organoids cultured for 7 days
Figure 3. Colon cancer organoids cultured for 6 days
Figure 4. Pancreatic cancer organoids cultured for 9 days
Figure 5. Ovarian cancer organoids cultured for 7 days
III.Troubleshooting
Q1: What concentration of Matrix Gel is recommended for organoid culture?
A1: Use full-strength Matrix Gel (typically 8–12 mg/mL) to support optimal 3D growth and maintain organoid morphology.
Q2: Can organoids be passaged in Matrix Gel?
A2: Yes. Organoids can be mechanically or enzymatically dissociated and re-embedded in fresh Matrix Gel for expansion.
Q3: How should Matrix Gel be handled during organoid preparation?
A3: Keep the gel and all tools on ice to prevent premature solidification. Once plated, allow the gel to polymerize at 37°C for 20–30 minutes before adding culture medium.
Q4: Are different types of Matrix Gel suitable for various organoid systems?
A4: Yes. Standard concentration Matrix Gel works for most epithelial organoids, while reduced growth factor formulations are ideal for systems requiring exogenous growth factor control.
Q5: What digestion solution should be used for tissue digestion, and what is the digestion duration?
A5: Tissue can be digested using either EDTA or collagenase. For hollow organs such as the small intestine and stomach, EDTA is suitable. Collagenase comes in different types with broad tissue applicability and may be combined with DNase. Digestion times vary significantly across tissues, ranging from several minutes to several hours.
Q6: Volumes of matrix gel and culture medium required per well for organoid culture in different plate formats?
A6: Commonly used plates for organoid culture include 24-well plates, where a 50 μL droplet of matrix gel is dispensed per well, overlaid with 500 μL of culture medium; 96-well plates use 10 μL of matrix gel per well, covered with 200 μL of medium; for 6-well plates, multiple 50 μL gel droplets can be seeded per well, with 2–3 mL of medium added to cover the droplets.
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